JPH08256771A - New protein having amidase activity and gene coding the same - Google Patents

Abstract

PURPOSE: To obtain the subject protein useful for producing the corresponding optically active organic acid from a racemic amide. CONSTITUTION: This new protein is obtained from culture of Comamonas acidovorans KPO-2772-4 strain (FERM P-14,842), etc., and has the following physical and chemical properties. (1) Acting on amides, catalyzing a reaction for hydrolyzing it into a carboxylic acid and ammonia and forming R-ketoprofen by enantiomer selective hydrolysis of at least ketoprofenamide (3-benzoyl-α- methylbenzeneacetamide). (2) Substrate-specifically acting on an aliphatic amide and especially strongly acting on n-capronamide. (3) Molecular weight: 54.0±2.0kDa (SDS-PAGE). (4) Optimum pH is at 8.5 to 10.0. Optimum temperature is at 35 deg.C. (5) The N-terminal amino acid sequence is PNADLHYWEAAELARKIRARDISPV.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel protein having an enantioselective amidase activity, a gene encoding the same, and a method for producing an organic acid from amides using a microorganism containing the gene, Comonas acidovovora.
ns) KPO-2771-4 strain (FERM P-14
842). Specifically, a novel protein having an activity of enantioselectively producing the corresponding organic acid from an amide and a gene encoding the same and a microorganism or a preparation thereof containing the gene are converted to a racemic amide. The present invention relates to a method of reacting to produce a corresponding optically active organic acid.

[0002]

BACKGROUND ART In order to hydrolyze amides and produce an organic acid, a chemical synthesis method is known in which an acid or an alkali is used as a reaction catalyst. In recent years, a microbial synthesis method using a microorganism containing amidase as a catalyst has come into use. This microbial method is widely used in the production of optically active organic acids because it can carry out the reaction at room temperature and atmospheric pressure and can also carry out the enantioselective reaction (Japanese Patent Laid-Open Publication No. Sho. 61-
88894, JP-A-62-55098, JP-A-1-309695, and JP-A-1-240199.
Japanese Patent Laid-Open No. 2-84198, Japanese Patent Laid-Open No. 5-50
7625, Japanese Patent Application Laid-Open No. 5-30992, Japanese Patent Application Laid-Open No. 5-76391, Japanese Patent Application Laid-Open No. 5-49498).

However, in the industrial use of this microbial method, in the stage of preparing a microbial catalyst, there are many factors depending on each microorganism, such as safety evaluation of the microbial catalyst and examination of an optimal culturing method for sufficiently enhancing the amidase activity. It is necessary to solve the problem and it may not always be easy to clear the cost aspect. On the other hand, as an improved method of such a microbial method, a method of enantioselectively producing a corresponding organic acid from an amide by examining the microbial catalyst itself, that is, making amidase in a more advantageous state by using a genetic engineering method, is examined. (Japanese Patent Laid-Open No. 4-218379 and Japanese Patent Laid-Open No. 5-233693).

[0004]

DISCLOSURE OF THE INVENTION The present invention is to search for a microorganism having a stronger amidase activity as compared with the conventional amidase activity, isolate and purify this amidase, and express the amidase using a genetic engineering technique. However, it is intended to provide a suitable technique for enantioselectively producing the corresponding organic acid from an amide.

[0005]

Means for Solving the Problems The inventors searched for and found a microorganism having a stronger amidase activity, and isolated and purified this amidase for the first time. Furthermore, the gene encoding this protein was also successfully isolated and expressed for the first time, and the present invention was completed. Next, the present invention will be described in detail.

The microorganism used in the present invention is searched for in the field soil of Saga Prefecture as one that can be assimilated and grown by using R-ketoprofenamide (R-3-benzoyl-α-methylbenzeneacetamide) as the only N source. However, Comamonas acidovora, which has been selected as having a strong amidase activity,
ns) KPO-2771-4 strain. This strain was deposited at the Institute of Biotechnology, Institute of Industrial Technology, deposit number FERM P-
Deposited as 14842. The mycological properties are shown below. Morphology: Bacillus 0.6-1.3 × 1.4-2.7 μm Gram stain; -Spore; -Mobility; + Growth state in broth agar plate culture; Round, smooth, glossy, white. Growth temperature; 37 ° C. +, 41 ° C.-, 45 ° C.-catalase; + oxidase; + glucose fermentation (OF test);-NO ₃ reduction; + indole production; -acid from glucose; -arginine dihydrolase; -urease; -Esculin hydrolysis; -Gelatin hydrolysis; -β-galactosidase; -Glucose assimilation; -Arabinose assimilation; -Mannose assimilation; -Mannitol assimilation; -N-acetylglucosamine assimilation; -Maltose assimilation; -Gluconic acid assimilation; -Capric acid assimilation; - assimilation adipic acid; + malate assimilation; + citrate assimilation; + phenylacetic acid assimilation; + cytochrome _{oxidase; + NO 3 -N 2; -} NO 2 -N 2; - growth at 3% sodium chloride; (+) Tween 80; + Alket of acetamide -Allantoin alkalization; + Tartaric acid alkalization; + Acid from fructose; + Acetic acid assimilation capacity; + In the above mycological properties, it is a gram-negative and motile rodent, and catalase activity Since it has oxidase activity, glucose OF test is negative, and fructose OF test is positive, it is judged to be a microorganism belonging to the genus Comamonas, and procarious (The PROK
ARYOTES, Second edition, 19
92, Balows A .; et. al. , Berli
n, Springer-Verlog) and Berjay's Manual of Systematic Bacteriology (Bergey's Manual of S).
ystematic Bacteriology, Vo
lume1, 1984, Sneat P. H. A. e
t. al. , Baltimore. Williamsa
ND Wilkins), the strain was identified as belonging to Comamonas acidborons belonging to the genus Comamonas, and the strain was identified as Comamonas acidborans KPO-2771-.
It was named 4 strains.

The culture of this strain can be carried out according to a known culture method. The culture conditions are usually liquid culture, but industrially, it is advantageous to perform deep aeration stirring culture. As the nutrient source of the medium, those usually used for culturing microorganisms can be widely used. The medium to be used may be one known as a nutrient source for general microorganisms, for example glucose, saccharose, lactose, molasses, glycerin, fumaric acid, citric acid, acetic acid, glutamic acid and other carbon sources such as ammonium sulfate, urea and ammonium chloride. And the like, organic nutrients such as meat extract, yeast extract, peptone and the like as nitrogen sources, inorganic nutrients such as vitamins, phosphoric acid, magnesium, potassium and iron can be used in appropriate combination. Further, isobutyronitrile, 2-azacyclononanone, N-methylcaprolactam and the like may be added as a substance that increases the amount of amidase activity in the microorganism. The pH of the medium is 5 to 10, and the culture temperature is 10 to 3.
It should be selected in the range of 8 ° C. The number of culture days may be in the range of 1 to 5 days until the target amidase content is maximized.

For the purification of the enzyme, a usual enzyme purification method can be used. The cells are collected from the culture-completed liquid by centrifugation or the like, and the cells are disrupted by a mechanical method such as ultrasonic treatment. The residue is removed by centrifugation to obtain the crude enzyme. next,
It is purified by ultracentrifugation fractionation, salting out, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, crystallization and the like.

The enzyme activity was measured as follows.
Ketoprofenamide 5 μmol, potassium phosphate buffer (pH 8.0) 10 μmol, bovine serum albumin 1 m
g and an appropriate amount of the enzyme solution were added and mixed so as to have a volume of 1.0 ml and reacted at 30 ° C. for 30 minutes, and then 4.0 ml of methanol was added to stop the reaction. The ketoprofen (3-benzoyl-α-methylbenzeneacetic acid) produced was measured by high performance liquid chromatography. The conditions are as follows: a column of Novapak ^™ Phenyl (Waters) at a flow rate of 254 nm, where a solvent obtained by mixing 0.05 M monopotassium phosphate (pH 3.5) and acetonitrile at 60:40 is eluted at 1 ml / min.
The absorbance was detected. In the examination of the substrate specificity and the like, the ammonia produced by the reaction was treated with J. Clin. P
ath. , 13 , 156 (1960). The amount of the enzyme that produces 1 μmol of ketoprofen or ammonia per minute was defined as 1 unit.

The optical purity of ketoprofen was determined by high performance liquid chromatography (conditions: a column of Sumichiral OA-2500S, eluting a methanol solvent containing 0.03 M ammonium acetate at 1.0 ml / min and detecting the absorbance at 254 nm). Measured. The homogeneity of the enzyme is S
DS-polyacrylamide gel electrophoresis was performed on a 4-12% gel using Tris-glycine buffer, J. Biol.
Chem. , 244 , 4406 (1969). One uniform protein band was observed, and the molecular weight of this protein was 5 compared with the known standard protein.
It was 4.0 ± 2.0 kDa.

The properties of the enzyme were as follows after uniform purification. Enzymatic action; acting as a catalyst for the reaction of acting on amides and hydrolyzing to carboxylic acid and ammonia. At least for ketoprofenamide, it exhibits a function as a catalyst for the reaction of enantioselective hydrolysis to produce R-ketoprofen. Substrate specificity: acts on aliphatic amides, but particularly strongly on n-capronamide. Molecular weight: The molecular weight was 54.0 ± 2.0 kDa by SDS-PAGE and filtration method, and it is a monomer composed of a single subunit. N-terminal amino acid sequence; A applying Edman degradation reaction
The N-terminal amino acid sequence is as follows as a result of application to a gas phase protein sequencer manufactured by Applied Biosystem and analysis of PTH amino acid by HPLC method. Pro-Asn-Ala-Asp-Leu-His-T
yr-Trp-Glu-Ala-Ala-Glu-Le
u-Ala-Arg-Lys-Ile-Arg-Ala
-Arg-Asp-Ile-Ser-Pro-Val- From this sequence, the Brevibacterium R312 and Rhodococcus amidase described in JP-A-4-218379 and the comamonas amidase described in JP-A-5-233693 are completely unidentified. The sequences are different, indicating that the protein of the present invention is completely novel. Optimum pH and optimum temperature; various buffers [-●-; potassium phosphate buffer (pH 5.5 to 8.0), -Δ-; Tris-hydrochloric acid buffer (pH 8.0 to 9.0), -O-; Glycine-NaOH buffer (pH 9.0 to 11.0)] was used to check the optimum pH, and as shown in FIG.
Is fairly wide and the best is around 8.5-10.0. Three
The hydrolytic activity became maximum around 5 ° C. Temperature stability: 75% or more of the activity remained even after being left at 28 ° C for 1 week in phosphate buffer solution pH 8.0.

Examples of the gene encoding the enzyme protein having the above properties include those having a base sequence as shown in SEQ ID NO: 3 in the sequence listing. The gene DNA fragment encoding the protein of the present invention can be obtained, for example, by the following method. As a DNA library containing a gene encoding the protein of the present invention, there are Comamonas acidborans (Com) isolated from soil.
amonas acidovorans) KPO-27
A plasmid library prepared by a conventional method using the chromosomal DNA prepared from the 71-4 strain (FERM P-14842) can be used.

The gene DNA of the present invention can be constructed by the following steps. (1) Chromosomal DNA was extracted from Comamonas acidoborans KPO-2771-4 strain and an appropriate restriction enzyme (for example, Sau3A) was extracted.
Partial decomposition with 1). (2) The DNA fragment obtained in (1) is incorporated into an appropriate vector to construct a chromosomal DNA library of Comamonas acidoborans. (3) A mixture of oligonucleotides having a DNA base sequence of a gene deduced from a part of the obtained N-terminal amino acid sequence of the purified amidase was synthesized, and this is called a DNA probe. (4) Label the DNA probe obtained in (3),
Hybridization is carried out with the library obtained in step 1, and clones showing homology with the probe are selected and separated. (5) Extract DNA from the clone selected in (4),
Southern hybridization is carried out using the above-mentioned probe to locate the gene of interest on a restriction enzyme fragment of an appropriate size, and it is positively subcloned into a mid vector. (6) Comamonas acidoborans KP incorporated into a vector using a subcloned plasmid
The nucleotide sequence of the DNA derived from strain O-2771-4 is determined. (7) The open reading frame of the amino acid sequence of amidase was found in the determined DNA base sequence, and D obtained in the above step was found.
Confirm that the entire coding region for amidase is present in the NA fragment.

In the above steps, the general operations necessary for handling DNA and Escherichia coli as a recombinant host are commonly performed by those skilled in the art, for example, Maniati.
s et al.'s experimental manual (T. Maniatis et a
l. , Molecular Cloning A La
laboratory Manual, Cold Spr
ing Harbor Laboratory 198
2, 1989). Commercially available products can be used for the enzymes and reagents used,
Unless otherwise specified, these objectives can be completely achieved by using the usage conditions specified for the product.

In the above (1), total DNA extraction from the bacterium can be performed by, for example, Saito et al. [Biochim. Bio
phys. Acta, 72, 619-629 (196
3)] can be performed according to the method. Examples of the vector in (2) above include EMBL3 and EMB.
Lambda phage vector such as L4 (STRATAGENE) or pHC79 (Bethesda Re)
search Laboratories <BRL>
Cosmid vectors such as those manufactured by K.K.) can be used. The mixture of oligonucleotides having the nucleotide sequence designated in (3) above can be synthesized by using a commercially available DNA synthesizer according to the procedure. For labeling in the above (4), any conventionally used radioisotope or a non-radioactive compound such as digoxigenin or biotin can be used, and labeling with digoxigenin is performed using a non-radio system DNA labeling system (DNA Labeling Kit). : Boehri
nger company) and follow the protocol attached thereto. A publicly known method can also be used for the method of determining the DNA base sequence in the above (6) [see, for example, literature: Sanger et al., Proc. N
at1. Acad. Sci. USA (1977), 7
4, 5463-5467].

In the following examples, unless otherwise specified, DNA fragments generated by digestion with restriction enzymes are separated and purified from an agarose gel by Gene Clean (GENE).
CLEAN ^{™ (} sold by Funakoshi Co., Ltd.) according to the attached protocol. Basic operations in subcloning, plasmid construction, etc. (ligation reaction between DNA fragments, transformation of Escherichia coli with hybrid plasmid generated by the reaction, transformation from the obtained transformant) The preparation of the plasmid and its analysis, etc.) were all carried out in accordance with the above Maniatis experimental manual.

Recombinant Escherichia coli carrying the amidase gene obtained by the above method was transformed into Escherichia coli (E
Sherichia coli) KP02 strain,
Deposited number FERM to Institute of Biotechnology, Institute of Biotechnology, AIST
Deposited as P-14841. Furthermore, by incorporating the gene obtained by the present invention into various expression plasmids for Escherichia coli [for example, pTrc99A (Pharmacia)], it becomes possible to construct a high amidase-expressing strain. Also, for other microorganisms, for example, bacteria belonging to the genus Brevibacterium, bacteria belonging to the genus Rhodococcus, bacteria belonging to the genus Bacillus, actinomycetes and yeasts, those for which a host-vector system has been established By using it, it becomes possible to create an amidase high expression strain by incorporating the obtained gene into a vector as in the case of E. coli. For example, in the case of a bacterium belonging to the genus Brevibacterium, Galzy et al. (FEMS Mic
robbiology Letters, 122, 129
~ 136 (1994)), in the case of a bacterium belonging to the genus Rhodococcus, Beppu et al. (Eur. J. Bioc).
hem. , 181, 563-570 (1989).

When culturing this KP02 strain,
The culture conditions may be selected in consideration of the nutritional physiological properties, and liquid culturing is usually carried out in most cases, but it is advantageous industrially to carry out deep aeration stirring culture. As a nutrient source for the culture, a carbon source, a nitrogen source, and an inorganic nutrient source used for culturing the microorganisms described above can be used in an appropriate combination.

The temperature of the culture can be appropriately changed within the range in which the microorganism grows and produces amidase, but in the case of Escherichia coli, it is preferably about 20 to 42 ° C. Although the culture conditions are slightly different depending on the conditions, the culture may be completed at an appropriate time in consideration of the time when the maximum yield of amidase is reached, and it is usually about 12 to 48 hours. Medium pH
Can be appropriately changed within the range where the bacterium grows and produces amidase, and the pH is preferably about 6-8.

The present invention also provides the following general formula (I)

[0021]

Embedded image

(In the formula, R ₁ represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocyclic group, and R ₂ represents a halogen atom, a hydroxy group or an alkyl group. However, the racemic amides represented by R ₁ and R ₂ are not the same, and the microorganism containing the gene described above or a preparation thereof is allowed to react with the following general formula (II )

[0023]

[Chemical 4]

A method for producing an optically active organic acid, characterized in that an optically active organic acid represented by the formula (wherein R ₁ and R ₂ are the same as above) is obtained. The reaction method of the present invention comprises a microorganism or its preparation and a compound of formula (I)
By contacting racemic amides represented by First, in the racemic amides represented by the general formula (I) in the present invention, for example, the alkyl group of the group R ₁ is, for example, a straight chain or branched chain having 1 to 10 carbon atoms and has an unsaturated bond. You may. Examples of the aryl group include a phenyl group and a naphthyl group.
Examples of the heterocyclic group include nitrogen, oxygen, and
Contains 1 to 3 of at least one sulfur and has 3 carbon atoms
Those consisting of a heterocycle composed of ~ 15 are preferred.
The substituent in the group R ₁ has, for example, 1 to 1 carbon atoms.
4, lower alkyl group, amino group, substituted amino group which may have lower alkyl group, hydroxy group, carboxyl group, thiol group, nitro group, appropriate substitution of halogen atom such as fluorine, chlorine, iodine, bromine, etc. Groups.

The group R ₂ is, for example, a halogen atom such as fluorine, chlorine, iodine or bromine, a hydroxy group or a linear or branched chain having 1 to 10 carbon atoms and may have an unsaturated bond. An alkyl group is mentioned. Specific compounds are preferably ketoprofenamide, ibuprofenamide [α-methyl-4- (2-methylpropyl) benzeneacetamide], pranoprofenamide [α-methyl-5H- (1) benzopyrano (2, 3-
b) Pyridine-7-acetamide], naproxenamide [(±) -6-methoxy-α-methyl-2-naphthaleneacetamide], mandelic acid amide [α-hydroxybenzeneacetamide], phenylbutyric acid amide, 2-chloropropionic acid Amide, β-phenyllactic acid amide, 4
Examples thereof include -chloro-α- (1-methylethyl) phenylacetic acid amide and 3-trifluoro-2-methylpropionic acid amide.

The optically active organic acid represented by the general formula (II) in the present invention is an acid obtained by enzymatically hydrolyzing an amino group from the amide represented by the general formula (I). The target compounds are compounds useful as medicines, agricultural chemicals, liquid crystals and intermediates thereof, or useful as optical resolving agents. In addition, the microorganism or the preparation thereof in the present invention specifically means a culture obtained by culturing a comamonas acidborans strain KPO-2771-4 strain or a transformant containing the gene, cells collected therefrom, or It shows a treated product of cells (for example, a crushed product of the cells or an enzyme separated and extracted from the cells), and further a product obtained by immobilizing the cells or the processed product of the cells on a carrier by an appropriate method.

As the reaction conditions in the present invention, the reaction medium may be water, a buffer solution or an aqueous medium mixed with an organic solvent, or a two-phase system of an organic solvent and an aqueous medium. The racemic amide represented by the formula (I) is added to the reaction medium as a solid or liquid as it is, or after dissolved in a suitable solvent. The addition concentration of the amide may be selected in the range of 0.01 to 70% by weight, and it may not be completely dissolved in the reaction medium. As the microorganism or its preparation used for amidase activity, the amount to be used may be appropriately determined based on the amidase activity, and may be appropriately changed depending on the concentration of amides as a raw material, for example, 0.01 to 20 units / ml. You can select within the range. Reaction temperature is 5 to 60 ° C, reaction p
H is in the range of 4 to 12, and reaction time is in the range of 1 to 200 hours. The amide may be supplemented continuously or intermittently.
The organic acid represented by the formula (II) thus produced may be continuously extracted out of the reaction system using a membrane or the like. The reaction may be stopped within a range in which the content of the optically active organic acid produced does not decrease, and is usually carried out until the reaction rate reaches the range of 10 to 60%.

Recovery of the optically active organic acid in the present invention is carried out as follows. After removing insoluble matters such as bacterial cells from the reaction-terminated liquid, the pH was adjusted to 7.5 to 10, and unreacted amides and the like were extracted and removed with a solvent such as n-butanol, benzene, diethyl ether, chloroform and the like. ,
The target substance is recovered by adjusting the pH to 2 to 4 and extracting with a solvent such as n-butanol, benzene, diethyl ether, chloroform and the like. Further, the target product is purified by treatment with silica gel, activated carbon or the like, crystallization and the like.

[0029]

EXAMPLES Next, the present invention will be described in more detail by way of examples, which should not be construed as limiting the invention thereto.

[0030]

Example 1 Production of R-ketoprofen by bacterial cell reaction Sodium fumarate 1%, yeast extract 1%, polypeptone 0.5%, dipotassium phosphate 0.2%, magnesium sulfate heptahydrate 0.02%, Ferrous sulfate heptahydrate 0.003
%, Sodium chloride 0.1%, isobutyronitrile 0.
Sterilized medium containing 1% and having a pH of 6.8 500 ml
In addition, the Komomonas acidborans KPO-2771-4 (FERM P-1484) previously cultured in the same medium was used.
2) The strain was inoculated with 2% and cultured with shaking at 28 ° C. for 2 days. After culturing, the bacterial cells were collected by centrifugation and suspended in an Erlenmeyer flask containing 160 ml of 0.01 M phosphate buffer (pH 8.0), followed by ketoprofenamide (3-benzoyl-α-methylbenzeneacetamide). ) Add 2g,
The reaction was carried out at 32 ° C with shaking.

After 30 hours, the reaction was completed, the cells were removed by centrifugation, the pH of the supernatant was adjusted to 11, and 200 ml of dichloromethane was added to extract and remove unreacted amide and the like. After adjusting the pH of the aqueous layer to 2 with hydrochloric acid,
The target substance was extracted by adding 200 ml of dichloromethane. Next, 10 g of activated carbon was added and sufficiently stirred, and then the activated carbon was removed by filtration. The liquid was concentrated and cooled to obtain crystals. When this was dried under reduced pressure at 50 ° C. for 1 day, 780 mg of R-(−)-ketoprofen was obtained.

This product was single in TLC chromatography and high performance liquid chromatography. The optical purity is 98.0% e.p. by specific optical rotation [α] _D ²⁵ = -41.6 ° (c = 1, ethanol) and optical purity measurement by high performance liquid chromatography. e. Became.

[0033]

Example 2 Purification of Amidase In the same culture as in Example 1, 2000 ml of Comamonas acidoborans KPO-2771-4 strain was cultured.
The cells are collected by centrifugation, washed with 0.1 mM potassium phosphate buffer [pH 8.0] (hereinafter, this buffer is abbreviated as KPB), and then suspended in 103 ml of KPB. This solution is subjected to ultrasonic treatment at 9 kHz for about 3
The cells were crushed for 0 minutes. Crushed cells are 15,000
The cells were removed by centrifugation at xg for 20 minutes to obtain a cell-free extract.

Ammonium sulfate was added to this so as to be 30%, and the supernatant was obtained by centrifugation. Ammonium sulfate was further added to this to 70% and the active fraction was obtained as a precipitate by centrifugation. After dissolving this precipitate in 50 ml of KPB, a sufficient amount of KPB
It was dialyzed at 30 to 70% ammonium sulfate. Then 10
Ultracentrifugation was performed at 20,000 xg for 2 hours, the supernatant was passed through DEAE-cellulose, and the enzyme was eluted with a KPB gradient containing KPB and 0.5 M sodium chloride. Collect active fractions and add 30% ammonium sulfate
Phenyl sepharose CL-
The enzyme was eluted with a gradient of KPB containing 30% ammonium sulfate and KPB containing 60% ethylene glycol by passing through a 4B column.

The active fractions were collected and dialyzed against KPB, then D
EAE-cellulose was passed through and the enzyme was eluted with a KPB gradient containing KPB and 0.1 M sodium chloride. The collected active fractions were concentrated by ultrafiltration (using YM10 membrane [Amicon Co.]) to obtain Sephacryl.
The amidase was purified by passing it through a column of S-400. The purification process is shown in Table 1.

[0036]

[Table 1]

[0037]

Example 3 Examination of Substrate Specificity The substrate specificity was examined using the amidase uniformly purified in Example 2. Amide compound 5 μmol, potassium phosphate buffer (pH 8.0) 10 μmol, bovine serum albumin 1 mg, 25 μl methanol, and 0.001 to 0.1 unit of amidase were added, mixed to 1.0 ml, and mixed at 30 ° C. The reaction was carried out for 30 minutes, and the activity was determined by quantifying the produced ammonia. Regarding the activity against various amide compounds, Table 2 shows the results of the relative activity when ketoprofenamide was set to 100%. As a result, it acts on fatty acid amides, especially n-capronamide.

[0038]

[Table 2]

[0039]

Example 4 Examination of optimum pH Example 2. The optimum pH was examined using amidase purified uniformly in. Ketoprofenamide 5 μmo
1, 10 μmol of each buffer, 1 mg of bovine serum albumin and 0.01 unit of amidase were added and mixed to make 1.0 ml, and the reaction was carried out at 30 ° C. for 30 minutes, and the activity was determined by quantifying the ketoprofen produced. I asked. The buffer solution is a potassium phosphate buffer solution having a pH of 5.5 to 8.0, p
H7.5-9.0 Tris-HCl buffer, pH 9.0-
A 10.5 glycine-NaOH buffer was used. The results are shown in Fig. 1, and the optimum pH is 8.5-10.0.
It was near.

[0040]

Example 5 Preparation of Gene Library of Comamonas acidoborans KPO-2771-4 Strain Soil Isolate Comamonas acidoborans KPO-2
The chromosomal DNA of 771-4 strain was prepared as follows.

This strain was used as the medium 100 in Example 1 above.
After inoculating and culturing in ml, the cells were collected, washed with water, and then suspended in 16 ml of TE [10 mM Tris-HCl (pH8.0), 10 mM EDTA], 16 mg of lysozyme and achromopeptidase 8 were added.
After adding KU and shaking at 37 ° C. for 1 hour, 0.4 ml of water containing 4 mg protease K was added and further shaking for 15 minutes. Next, 2 ml of 10% SDS was added, and the mixture was shaken at 37 ° C. for 1 hour and then incubated at 55 ° C. for another 10 minutes. Then an equal amount of TE saturated phenol was added and shaken for 10 minutes. After centrifugation, remove the upper layer, add an equal amount of TE saturated phenol / chloroform, and shake for 5 minutes (hereinafter,
This operation is called phenol treatment), after adding 2.5 times the amount of ethanol to the upper layer after centrifugation, DNA is then added with a glass rod.
Was taken up and dehydrated with 70% ethanol. Next is DNA
Was dissolved in 2 ml of TE buffer, then at 100 ° C for 1
The RNase A aqueous solution that had been heat-treated for 0 minutes was added to a final concentration of
ug / ml was added and the mixture was incubated at 37 ° C for 20 minutes. Then 1.2 ml of PEG-NaCl [20% PEG 600
[0,2.5 M NaCl] was added and the mixture was allowed to stand at 4 ° C. for 2 hours, then centrifuged and the DNA was recovered by ethanol precipitation. As a result, 1.0
mg of chromosomal DNA was obtained.

The resulting chromosomal DNA was partially digested with Sau 3A (Takara Shuzo) and then EMBL3 CLONIGK
Bam in λEMBL3 using IT (STRATAGENE)
It was incorporated into the HI site. Next, this DNA is GIGAPACK II
Using PACKAGING EXTRACT (manufactured by STRATAGENE), E. coli was packaged in λ phage according to the protocol. c
Infection with oli LE392 was found to contain 6 × 10 ⁵ recombinant phages per ml.

[0043]

Example 6 Screening of Clones Containing DNA Fragment Encoding Amidase Protein Based on the N-terminal amino acid sequence determined from the purified amidase, the synthetic mixDNA KP01 (2) shown in SEQ ID NOs: 1 and 2 of the Sequence Listing.
0 mer, 96 mix) and KP02 (20 mer, 25
6 mix) was chemically synthesized, and 400 pmol of each was mixed with non-radio system DNA labeling system (DNA Labeling Ki).
, manufactured by Boehringer) according to the protocol attached thereto, and digoxigenin-labeled deoxyuridine-3 is used.
-Labeled with phosphoric acid (DIG-dUTP). Hereinafter, these probes will be referred to as KP01 and KP02-probes.

A portion of the phage solution (gene library) prepared in Example 5 above was infected with LE392 to form 4 × 10 ³ plaques on 4 plates. These plaques were transferred to a filter (Hybond-N, manufactured by Amersham), subjected to alkali denaturation and neutralization treatment, and then UV irradiation
Was immobilized and then hybridized with the KP01 and KP02-probes obtained above. Hybridization was performed with 5 × SSC, 0.02% SDS, 1% blocking agent (Blocking Reagent, manufactured by Boehringer),
0.1% N-lauroyl sarcosine and final concentration 10 pm
It was carried out overnight at 50 ° C. with a solution containing KPO-probe at ol / ml.

Next, the filter was washed twice in a 6 × SSC solution containing 0.1% SDS at room temperature for 5 minutes each time, and the KP01-probe was added to the buffer with 54, 58 and the same buffer.
KP02-probe was 56, 60, 6 at 62, 66 ° C.
It was washed once at 4 and 68 ° C. for 5 minutes each. Next, a detection kit (DIG Luminescent) using an anti-digoxigenin antibody labeled with alkaline phosphatase and the luminescent substrate AMPPD
Detection Kit, manufactured by Boehringer) was used to detect clones hybridized with KP01 and KP02-probes under the most severe conditions according to the attached protocol.

As a result, 5 positive spots were found. The phage clones corresponding to these spots were designated as λ-KP01, λ-KP02, λ-KP03 and λ-KP.
04 and λ-KP05, respectively. From the obtained 5 phage clones, the method described in Grossberger (Reference: Nucleic Acids, Research (1987) 15,6737)
DNA was extracted according to. Then, this lambda DNA was digested with BglII, subjected to agarose gel electrophoresis, and then subjected to Southern hybridization using a KP01-probe (for a method, see Reference: Southern,
[J. Mol. Biol. (1975) 98, 503-517]. The filter after hybridization (High Bond N)
The same operation as described above was carried out except that the washing was carried out at 54 ° C. As a result, the above clone λ-KP0
It was found that the 2.7 Kb BglII fragment present in 4 hybridizes with the probe.

[0047]

Example 7 Subcloning of DNA Fragment and Determination of Nucleotide Sequence pKP02 was obtained by subcloning the 2.7 Kb BglII fragment obtained in Example 6 into the BamHI site of the plasmid vector pUC118.

Next, pKP02 was cleaved with various restriction enzymes and analyzed by agarose gel electrophoresis. As a result, the restriction enzyme map of 2.7 Kb insert shown in FIG. 2 was obtained. The entire base sequence of the BglII-XhoI fragment in the above insert was determined based on the method of Sanger et al. As a result, the sequence of the translation product expected to be the nucleotide sequence as shown in SEQ ID NO: 3 in the sequence listing was obtained. The sequence consisting of 25 amino acids following methionine in the translation product sequence was completely in agreement with the N-terminal amino acid sequence determined from the purified protein. It was also confirmed that this enzyme is composed of 473 amino acids. The E. coli KP02 strain having pKP02 had a clear amidase activity as compared with Escherichia coli having a vector plasmid that did not retain the insert. Therefore, it was confirmed that the amidase gene was present in this insert. It was

[0049]

Example 8 Production of R-ketoprofen using Escherichia coli KP02 strain having pKPO2 Meat extract 0.3%, peptone 1%, sodium chloride 0.5%, isopropyl-β
2% of the KP02 strain previously cultivated in the same medium was inoculated into 500 ml of a sterilizing medium containing 1 mM (separated) of -D-(-)-thiogalactopyranoside and having a pH of 7.0, and 3
The cells were cultured at 7 ° C for 1 day with shaking. After culturing, the bacterial cells were collected by centrifugation, and this was added to 0.01M phosphate buffer (pH 7.5).
After suspending in an Erlenmeyer flask containing 20 ml, 200 mg of ketoprofenamide was added and reacted at 32 ° C. for 40 hours. The reaction-terminated liquid was sterilized and then purified in the same manner as in Example 1 to obtain 65 mg of R-(-)-ketoprofen. High-performance liquid chromatography has a purity of 99.5% and an optical purity of 99% e. e. Met.

[0050]

Industrial Applicability According to the present invention, a novel protein having amidase activity and a novel gene encoding this protein were obtained. It is possible to industrially advantageously produce an optically active organic acid by performing enantioselective hydrolysis from a racemic amide compound by allowing a microorganism containing this novel amidase and this gene or a preparation thereof to act. Became.

[Brief description of drawings]

FIG. 1 shows the optimum pH curve of the amidase of the present invention.

FIG. 2 is a diagram showing restriction enzyme sites of the amidase gene of the present invention.

[Sequence list]

 SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GAYYTNCAYT AYTGGGARGC

[Sequence list]

 SEQ ID NO: 2 Sequence length: 20 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence TAYTGGGARC CNGCNGARYT

[Sequence list]

SEQ ID NO: 3 Sequence length: 2438 Sequence type: Nucleic acid Number of strands: Double strand Topology-: Linear Sequence type: genomic DNA Origin organism name: Comamonas acidv
orans) Strain name: KPO-2771-4 Sequence characteristics Location: 485-1906 Method by which the characteristics were determined: E Characteristic symbol: CDS sequence AGATCTTCTT TGATGTCCGC ATCAATCCGC GCACCAGCCC GGCCGACGTC AAGGCCCAGT 60 TCGCGCGACGCGATCGAGGCTCGAGCGCGAGCTC CGCCGTCCAA CTGGATCGTC CAGTCGGCCA 180 GGCGCGGATG GGAGCATATC GAAGCGCGCC CGCATGGCAT TCCCGAGCTG CTGGGCGGAC 240 AGACCGACGG CGCGGCCCTG CGCCGCTTCG GCATTCCAAC CGCACGCATC GGCTGGCCAT 300 GGCCGGCGCA GGGTTCGCCC GAAAGCGTGG CCGAAGGCCT GGGTGGCATG GGTGCCACCT 360 ATGTCCCGGA CCTGCTGCCT TGCGCGCGCA AGATCATGTA CGCCGTGATC GATACGCTGA 420 CGCGCAGACG TGCGGAACTC GGCTTGCGCT GAGGACTCCC GACAAACTTC CCGACCCATT 480 GCTT ATG CCG AAC GCA GAC CTC CAC TAC TGG GAA GCC GCG GAG CTT GCA 529 Met Pro Asn Ala Asp Leu His Tyr Trp Glu Ala Ala Glu Leu Ala 1 5 10 15 CGC AAG ATC CGC GCG CGC GAC ATC TCG CCT GTA GAA GTT ACC GAA GCC 577 Arg Lys Ile Arg Ala Arg Asp Ile Ser Pro Val Glu Val Thr Glu Ala 20 25 30 ATC CTT GAA CGA ATC TCC GAC CTC GAT CCG GCG CTG CAC AGC TAC GCC 625 Ile Leu Glu Arg Ile Ser Asp Leu Asp Pro Ala Leu His Ser Tyr Ala 35 40 45 CTG GTC CTG CCT GAA CAG GCG CTG GAA CAG GCA CGC GAC GCC GAA CGG 673 Leu Val Leu Pro Glu Gln Ala Leu Glu Gln Ala Arg Asp Ala Glu Arg 50 55 60 GCG CTG ATG CAC GGC GGC CCG CTC GGC GCA CTG CAC GGC GTG CCG GTG 721 Ala Leu Met His Gly Gly Pro Leu Gly Ala Leu His Gly Val Pro Val 65 70 75 GCG GTC AAG GAC CTC TGC TGG CTG GCC GGA TCG CCA ACC GCC GCC GGC 769 Ala Val Lys Asp Leu Cys Trp Leu Ala Gly Ser Pro Thr Ala Ala Gly 80 85 90 95 ACC ACG ATT CAC AAA GAC TTT ATC CCG ACG GAA GAT GCC ACC GTC GTG 817 Thr Thr Ile His Lys Asp Phe Ile Pro Thr Glu Asp Ala Thr Val Val 100 105 110 CGC AAG CTG CGG GAT GCC GGC GCC ATC GTG CTG GGC AAG CTG CAG CTG 865 Arg Lys Leu Arg Asp Ala Gly Ala Ile Val Leu Gly Lys Leu Gln Leu 115 120 125 ACC GAA GGC GCC TTC GCC ACC CAT CAC CCG AGC ATA CCG GAG CCG GTC 913 Thr Glu Gly Ala Phe Ala Thr His His Pro Ser Ile Pro Glu Pro Val 130 135 140 AAT CCC TGG CAC CCC AAC CAC TGG GCC GGC GCC TCT TCC AGC GGC TCC 961 Asn Pro Trp His Pro Asn His Trp Ala Gly Ala Ser Ser Ser Gly Ser 145 150 155 GGT GTC GCC ACG GCC GCC GGG CTT TGC TAC GCA TCG ATC GGC ACG GAC 1009 Gly Val Ala Thr Ala Ala Gly Leu Cys Tyr Ala Ser Ile Gly Thr Asp 160 165 170 175 ACC GGC GGC TCG ATC CGC TTC CCG GCG GCA GCC AAC GGC CTG ACC GGC 1057 Thr Gly Gly Ser Ile Arg Phe Pro Ala Ala Ala Asn Gly Leu Thr Gly 180 185 190 ATC AAG CCG ACG TGG GGC CGG GTC AGC CGC CAC GGA GTA TTC GAG CTC 1105 Ile Lys Pro Thr Trp Gly Arg Val Ser Arg His Gly Val Phe Glu Leu 195 200 205 GGC GCG AGC CTT GAC CAT GTG GGT CCG ATC ACA CGC AGC GCA GCC GAT 1153 Gly Ala Ser Leu Asp His Val Gly Pro Ile Thr Arg Ser Ala Ala Asp 210 215 220 GCC GCG CTG ATG CTA GGG ACC ATT GCC GGC CAC GAT CCG CTG GAT CCG 1201 Ala Ala Leu Met Leu Gly Thr Ile Ala Gly His Asp Pro Leu Asp Pro 225 230 235 ACT TCG CTG CCA CCC TCG CAC CTT GAT TTA CCT GCC GAG AGG AGC GAC 1249 Thr Ser Leu Pro Pro Ser His Leu Asp Leu Pr o Ala Glu Arg Ser Asp 240 245 250 255 CTG CGC GGT CTG CGC ATC GGT GTC GAC GTG CAA TGG AAC AGC GAT GGC 1297 Leu Arg Gly Leu Arg Ile Gly Val Asp Val Gln Trp Asn Ser Asp Gly 260 265 270 ACG GAT GAC GTC ATC AGC CAG GCC ATC GAC CGG GCC ATC GAC GTG CTC 1345 Thr Asp Asp Val Ile Ser Gln Ala Ile Asp Arg Ala Ile Asp Val Leu 275 280 285 AAG GCG CTT GAC GGG CAG GTC CAG GAG GTT CGC TTT CCC GCA GTG CGC 1393 Lys Ala Leu Asp Gly Gln Val Gln Glu Val Arg Phe Pro Ala Val Arg 290 295 300 ACC GTG GTC GAC GAA TGG GAA ATC AAC TGC GGC GTC GAA GTC GCC GTG 1441 Thr Val Val Asp Glu Trp Glu Ile Asn Cys Gly Val Glu Val Ala Val 305 310 315 GCG CAT GCG GGG ACG TTC CCG CGC CTG TCG GCG GAA TAT GGG CCA GCG 1489 Ala His Ala Gly Thr Phe Pro Arg Leu Ser Ala Glu Tyr Gly Pro Ala 320 325 330 335 CTG ACC CGG CTG ATC GAG ATT GGT CGC AAT ACC AGC GGC ATG CGC TAT 1537 Leu Thr Arg Leu Ile Glu Ile Gly Arg Asn Thr Ser Gly Met Arg Tyr 340 345 345 350 CAG CAA GCC TTG CTG AAC CGT GCA GCT TTC CGT GGC AAG GTC AAC AAG 1585 Gln Gln Ala Leu Leu Asn Arg Ala Ala Phe Arg Gly Lys Val Asn Lys 355 360 365 CTG ATG CAG GAC ATC GAT CTG CTG GTC GTC CCG GTA CAG CCT TTC GCC 1633 Leu Met Gln Asp Ile Asp Leu Leu Val Val Pro Val Gln Pro Phe Ala 370 375 380 GCA CCG ACG CAT GAA CAG CTG GGT GCG CTG GCG CAA GAC CCG GAA CTG 1681 Ala Pro Thr His Glu Gln Leu Gly Ala Leu Ala Gln Asp Pro Glu Leu 385 390 395 AAC AGC CGG CTG ATC CAG TTC ACG GCG CCG TTC AAC TCC ACC GGG CAT 1729 Asn Ser Arg Leu Ile Gln Phe Thr Ala Pro Phe Asn Ser Thr Gly His 400 405 410 415 CCC GCG ATC ACG CTG CCA TGT GGA TTC ACC GCG GGC GGC ATG CCG ATC 1777 Pro Ala Ile Thr Leu Pro Cys Gly Phe Thr Ala Gly Gly Met Pro Ile 420 425 430 GCA TTC CAG CTG GTG GCG CGC CAT GGT GAG GAA GCA CTG CTG TGC CGA 1825 Ala Phe Gln Leu Val Ala Arg His Gly Glu Glu Ala Leu Leu Cys Arg 435 440 445 GCC GGC ATG GCA TTC CAG CAG GCC ACC GAC TGG CAC CGC TAC CAT CCG 1873 Ala Gly Met Ala Phe Gln Gln Ala Thr Asp Trp His Arg Tyr His Pro 450 455 460 GAG ATT GGC GTC AAG CGC GCT GAC GCT GCG TGATGAGATC AGGCCGGGCA 1923 Gl u Ile Gly Val Lys Arg Ala Asp Ala Ala 465 470 ATGCCGGCCT GATTTCATTC CTGCCCGTCT TCCCGCAACG GTGTGATCGT CGCCGAATCG 1983 CGCGCGGCTT CCTCATAGAT GCCGGTCAGC AGCGAAAGGA AGCGCGATGT CCTTTCAAAT 2043 TTCTCCGACA CCTCGCTGAT TTCCTCCAGC CGCTTCACCA GGATCTCGGC CCTGACCTTG 2103 TAATACTCGT CCGTCAGCCG GACTCCCAGT TCACTGACGG TGTACGCAAA GGTCTTGCTG 2163 TTCTTTTCTC GCTGCTTTTC GATCAGACCG GCCGCTTCCA GCTTGCGCAA GCTGTACTGA 2223 ATATTCGGGA TATCGTCGCG GTTCATCAGG CGCGCAATGG TGGCACTATT CTTCGGCCGG 2283 TCCTGCATCC TGATGACGTG GAGAATGACG TGCTCCTCGT ACTTCATATC CACGTCGGCG 2343 GCAATCGTCA CCATCCCGGT GGACAACACG AACCTGGAAA ACGATTCATG GAACCGGATC 2403 AAGGCCCACT CGAACTCGGT GG

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12P 41/00 9162-4B C12N 15/00 ZNAZ // (C12N 9/80 C12R 1:01) ( C12N 9/80 C12R 1:19) (C12N 1/20 C12R 1:01) (C12N 15/00 ZNA C12R 1:01) (C12P 7/40 C12R 1:01) (C12P 41/00 C12R 1:01)

Claims (5)

[Claims] 1. A novel protein having the following physicochemical properties. Action It acts on amides and functions as a catalyst for the reaction of hydrolyzing carboxylic acids and ammonia. At least ketoprofenamide (3-benzoyl-α-methylbenzeneacetamide) exhibits a function as a catalyst for the reaction of enantioselective hydrolysis to produce R-ketoprofen. Substrate specificity It acts on aliphatic amides, but particularly strongly on n-capronamide. Molecular weight Molecular weight is 54.0 ± 2.0 kDa (SDS-PAGE)
And is composed of a single subunit. N-terminal amino acid sequence Pro-Asn-Ala-Asp-Leu-His-T
yr-Trp-Glu-Ala-Ala-Glu-Le
u-Ala-Arg-Lys-Ile-Arg-Ala
-Arg-Asp-Ile-Ser-Pro-Val- Optimum pH and Optimum Temperature Hydrolysis action is optimum at pH 8.5-10.0 and 35 ° C. Temperature stability 75% activity even if left at pH8.0 and 28 ℃ for 1 week
The above remains. 2. An amino acid sequence 2 to 47 in SEQ ID NO: 3 of the sequence listing, which encodes the protein according to claim 1.
A gene represented by the base encoding 3. 3. The base is 4 in SEQ ID NO: 3 in the sequence listing.
The gene according to claim 2, which is 88 to 1903. 4. The following general formula (I): (In the formula, R ₁ represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocyclic group, and R ₂ represents a halogen atom, a hydroxy group, or an alkyl group. R ₁ and R ₂ are not the same), and the microorganism containing the gene according to claim 2 or a preparation thereof is allowed to act on the racemic amide represented by the following general formula (II) 2] (In the formula, R ₁ and R ₂ are the same as above.) A method for producing an optically active organic acid, which comprises obtaining an optically active acid. 5. A Combomonas acidbolans (Coma)
monas acidovorans) KPO-27
71-4 strain (FERM P-14842).